IL8 Blocking EMT Pathway and Overcoming Cancer Stem Cells

ABSTRACT

Compositions, methods, and uses of recombinant IL-8 antibody, fragment thereof or single chain variable fragment (scFv) having high affinity to IL-8 to target tumor-expressed or endogenous IL-8 are presented. Preferably, the recombinant IL-8 antibody or scFv fragment includes a V H  segment comprising a first amino acid sequence selected from SEQ ID NO. 1-15, 31-32, and/or a V L  segment comprising a second amino acid sequence selected from SEQ ID NO. 16-30, 33-34. The recombinant IL-8 antibody or scFv fragment can be formulated as pharmaceutical compositions to administer to a patient having a tumor to reduce metastasis of the tumor, reduce immune suppression in the tumor microenvironment or reduce Th2 mediated immune response.

This application is a divisional application of allowed with the U.S.patent application Ser. No. 16/174,040, which was filed Oct. 29, 2018,and which claims priority to our with the U.S. Provisional patentapplication Ser. No. 62/580,232, which was filed Nov. 1, 2017.

FIELD OF THE INVENTION

The field of the invention is compositions, methods, and use ofrecombinant scFv or antibody to interleukin-8 (IL-8) for treating apatient having a tumor to reduce metastasis of the tumor or to reduceimmune suppression in the tumor microenvironment.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Interleukin-8 (IL-8), also known as neutrophil chemotactic factor, ismainly produced by macrophages and epithelial cells, and has known toplay a key role in immune system by recruiting immune cells (e.g.,neutrophils) to the infection site. Recently, interest has been growingon identifying the relationship between the tumor-produced IL-8 anddevelopment of the tumor, especially in tumor metastasis and mechanismof immune-suppression in the tumor microenvironment. For example,several studies found that tumor-produced IL-8 is highly expressed inmetastatic tumor cells and indeed, is capable of induceepithelial-mesenchymal transition of the tumor cells to producetumor-initiating cells (e.g., tumor stem cells). Other studies showedthat tumor-produced IL-8 attracts Myeloid-Derived Suppressor Cells(MDSC), which can provide immune-suppressive microenvironment around thetumor by interfering T-cell mediated immune response in the tumormicroenvironment.

In order to mitigating the effect of IL-8 in tumor development, effortshad been put to neutralize tumor-expressed or endogenous IL-8 byproviding human or humanized antibody against IL-8, anti-senseoligonucleotides or microRNA against IL-8. For example, U.S. Pat. Pub.No. 2003/0068319 to Bar-Eli discloses inhibition of angiogenesis andmetastasis of tumor by fully humanized and isolated monoclonal orpolyclonal IL-8. In another example, U.S. Pat. No. 5,849,903 toPetrzkowski teaches 20 base pair-length anti-sense oligonucleotideagainst IL-8 effective to reduce growth of melanoma or lung carcinoma.However, the effect of anti-sense oligonucleotides or microRNA may varydepending on the type of tumor, and the delivery method of suchcompositions. Also, isolated human or humanized antibody may not beeffective in some tumor microenvironment where the isolated human orhumanized antibodies are cleaved by endogenous metalloprotease in thetumor microenvironment.

Thus, even though several approaches of inhibiting expression oractivity of IL-8 in tumor microenvironment have been studied, targetingIL-8 using recombinant IL-8 antibody or single chain variable fragment(scFv) with amino acid sequences of high affinity to IL-8 has remainedlargely unexplored. Therefore, there is still a need for improvedcompositions, methods for and uses of recombinant IL-8 antibody orsingle chain variable fragment (scFv) to target tumor-expressed orendogenous IL-8 in the tumor microenvironment to promote effect ofcancer therapy.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to various compositions of,methods for, and uses of a recombinant IL-8 antibody, single chainvariable fragment (scFv), or other portion of an antibody (includingfusion products, especially in a TxM) with high affinity to IL-8 totarget tumor-expressed or endogenous IL-8 and neutralize the effect ofIL-8 in promoting EMT (epithelial-mesenchymal transition) of a tumorcell, and/or immune-suppression in the tumor microenvironment.Consequently, the scFv peptides may be coupled to a protein having IL-15activity and/or to a protein having IL-15 receptor alpha activity to soform a TxM molecule.

Thus, one aspect of the subject matter includes single chain variablefragment (scFv) peptide. The scFv peptide includes a V_(H) segmentcomprising a first amino acid sequence and/or a V_(L) segment comprisinga second amino acid sequence. The first and second amino acid sequencesare selected from SEQ ID NO. 1-15, 31-32 or SEQ ID NO. 16-30, 33-34,respectively.

In another aspect of the inventive subject matter, the inventorscontemplate a pharmaceutical composition for treating a patient having acancer. The pharmaceutical composition includes single chain variablefragment (scFv) having includes a V_(H) segment comprising a first aminoacid sequence and/or a V_(L) segment comprising a second amino acidsequence. The first and second amino acid sequences are selected fromSEQ ID NO. 1-15, 31-32 or SEQ ID NO. 16-30, 33-34, respectively.Preferably, the scFv peptide is present in the pharmacologicallyacceptable carrier.

Still another aspect of inventive subject matter is directed towards arecombinant nucleic acid. The recombinant nucleic acid includes a firstnucleic acid segment encoding a V_(H) segment having a first amino acidsequence selected from the group consisting of SEQ ID NO. 1-15, 31-32and/or a second nucleic acid segment encoding a V_(L) segment having asecond amino acid sequence selected from the group consisting of SEQ IDNO. 16-30, 33-34. Optionally, the first and second segments are presentin a same reading frame.

In still another aspect of the inventive subject matter, the inventorscontemplate a recombinant isolated antibody or fragment thereof Therecombinant isolated antibody or fragment thereof includes a V_(H)domain and/or a V_(L) domain having respective first and second aminoacid sequences. Preferably, the first amino acid sequence is at least95% identical to a third amino acid sequence selected from the groupconsisting of SEQ ID NO. 1-15, 31-32, and the second amino acid sequenceis at least 95% identical to a third amino acid sequence selected fromthe group consisting of SEQ ID NO. 16-30, 33-34.

In still another aspect of the inventive subject matter, the inventorsalso contemplate a method of reducing an IL-8 effect in a tissue. Inthis method, a single chain variable fragment (scFv) having includes aV_(H) segment comprising a first amino acid sequence and/or a V_(L)segment is provided. Preferably, the first amino acid sequence is atleast 95% identical to a third amino acid sequence selected from thegroup consisting of SEQ ID NO. 1-15, 31-32, and the second amino acidsequence is at least 95% identical to a third amino acid sequenceselected from the group consisting of SEQ ID NO. 16-30, 33-34. Themethod continues with a step of treating the tissue with the scFvpeptide in a dose and schedule effective to reduce the IL-8 effect inthe tissue.

In still another aspect of the inventive subject matter, the inventorscontemplate a method of treating a patient having a tumor. In thismethod, a pharmaceutical composition including a single chain variablefragment (scFv) having a V_(H) segment comprising a first amino acidsequence and/or a V_(L) segment is provided. Preferably, the first aminoacid sequence is at least 95% identical to a third amino acid sequenceselected from the group consisting of SEQ ID NO. 1-15, 31-32, and thesecond amino acid sequence is at least 95% identical to a third aminoacid sequence selected from the group consisting of SEQ ID NO. 16-30,33-34. The pharmaceutical composition is administered to the patient ina dose and a schedule effective to treat the tumor.

In still another aspect of the inventive subject matter, the inventorscontemplate a method of reducing immune suppression in a patient havinga tumor. In this method, a pharmaceutical composition including a singlechain variable fragment (scFv) having a V_(H) segment comprising a firstamino acid sequence and/or a V_(L) segment is provided. Preferably, thefirst amino acid sequence is at least 95% identical to a third aminoacid sequence selected from the group consisting of SEQ ID NO. 1-15,31-32, and the second amino acid sequence is at least 95% identical to athird amino acid sequence selected from the group consisting of SEQ IDNO. 16-30, 33-34. The pharmaceutical composition is administered to thepatient in a dose and a schedule effective to reduce presence ofmyeloid-derived suppressor cells in a tumor microenvironment.

In still another aspect of the inventive subject matter, the inventorscontemplate a method of reducing Th-2 mediated immune response in apatient having a tumor. In this method, a pharmaceutical compositionincluding a single chain variable fragment (scFv) having a V_(H) segmentcomprising a first amino acid sequence and/or a V_(L) segment isprovided. Preferably, the first amino acid sequence is at least 95%identical to a third amino acid sequence selected from the groupconsisting of SEQ ID NO. 1-15, 31-32, and the second amino acid sequenceis at least 95% identical to a third amino acid sequence selected fromthe group consisting of SEQ ID NO. 16-30, 33-34. The pharmaceuticalcomposition is administered to the patient in a dose and a scheduleeffective to reduce Th-2 mediated immune response in the patient.

In still another aspect of the inventive subject matter, the inventorscontemplate a method of reducing epithelial-mesenchymal transition of atumor cell in a patient. In this method, a pharmaceutical compositionincluding a single chain variable fragment (scFv) having a V_(H) segmentcomprising a first amino acid sequence and/or a V_(L) segment isprovided. Preferably, the first amino acid sequence is at least 95%identical to a third amino acid sequence selected from the groupconsisting of SEQ ID NO. 1-15, 31-32, and the second amino acid sequenceis at least 95% identical to a third amino acid sequence selected fromthe group consisting of SEQ ID NO. 16-30, 33-34. The pharmaceuticalcomposition is administered to the patient in a dose and a scheduleeffective to reduce epithelial-mesenchymal transition of the tumor cell.

In still another aspect of the inventive subject matter, the inventorscontemplate use of the scFv peptide described above for reducing Th-2mediated immune response in a patient having a tumor. Also, theinventors contemplate use of the scFv peptide described above forreducing epithelial-mesenchymal transition of a tumor cell in a patient.In addition, the inventors further contemplate use of the scFv peptidedescribed above for reducing metastasis of a tumor in a patient havingthe tumor.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A shows a graph of binding kinetics between one of scFv peptide(43-12) with IL-8 or IL-8 orthologs or paralogs.

FIG. 1B shows a graph of binding kinetics between one of scFv peptide(49-31) with IL-8 or IL-8 orthologs or paralogs.

FIGS. 2A-D show graphs of neutrophil transmigration upon IL-8 treatment(2A, 2C) and with scFv peptide co-treatment with IL-8 (2B, 2D).

FIG. 3 shows a graph of neutrophil transmigration upon IL-8 treatment(CTRL) and with scFv peptide co-treatment with IL-8 (43-2, 43-12).

FIGS. 4A-C show graphs of binding kinetics between one of scFv peptide(43-12) with IL-8 under specific pH conditions (4A), and bindingkinetics for selected affinity maturated variant scFv peptides (43-12aand 43-12b) under specific pH conditions.

DETAILED DESCRIPTION

The inventors now discovered that various IL-8 mediated effects,including tumor development, particularly IL-8 mediated tumor metastasisthrough epithelial-mesenchymal transition pathway, can be significantlyreduced or even abrogated by mitigating the IL-8 effect. Such mitigatingeffect can be achieved by trapping or otherwise binding IL-8 in thetumor microenvironment using a recombinant IL-8 antibody or fragmentsthereof (e.g., scFv fragment against IL-8 with high affinity againstIL-8).

To that end, the inventors discovered that various recombinant antibodyor fragments thereof such as a scFv with high affinity against IL-8 canbe generated such that recombinant antibody or fragments thereof, orscFv fragments can trap tumor-generated or endogenous IL-8 in the tumormicroenvironment. Binding or entrapment of IL-8 from the tumormicroenvironment is thought to decrease the IL-8 effect to the tumorcells triggering metastasis of the tumor cells. Also, binding orentrapment of IL-8 can reduce the IL-8 effect of increasing immunesuppression in the tumor microenvironment via accumulatingmyeloid-derived suppressor cells (MDSC). Further, binding or entrapmentof IL-8 can reduce the IL-8 effect of increasing Th2-mediated immuneresponse in the tumor microenvironment, which can contribute torecruitment of myeloid-derived suppressor cells in the tumormicroenvironment. While not wishing to be bound by any specific theoryor hypothesis, it is contemplated that IL-8 bund to an antibody orfragment will no longer be able to exert its biological signalingfunction, possibly due to steric effects. Moreover, where binding ismediated on a particle or other surface to so entrap the IL-8, IL-8concentrations needed for signaling may be reduced and so reduce orabrogate IL-8 effects. Thus, the terms binding and entrapment are usedinterchangeably herein.

As used herein, the term “tumor” refers to, and is interchangeably usedwith one or more cancer cells, cancer tissues, malignant tumor cells, ormalignant tumor tissue, that can be placed or found in one or moreanatomical locations in a human body.

As used herein, the term “bind” refers to, and can be interchangeablyused with a term “recognize” and/or “detect”, an interaction between twomolecules with a high affinity with a K_(D) of equal or less than 10⁻³M, 10⁻⁴M , 10⁻⁵M , 10⁻⁶M, or equal or less than 10⁻⁷M.

As used herein, the term “provide” or “providing” refers to and includesany acts of manufacturing, generating, placing, enabling to use, ormaking ready to use.

In one exemplary and especially preferred aspect of the inventivesubject matter, the inventors contemplate a single chain variablefragment (scFv) peptide having a V_(H) segment and a V_(L) segment thatbinds to IL-8. While it is contemplated that the peptide sequence ofscFv can be any suitable sequences providing desirable binding affinityto IL-8, the inventors found that the scFv peptide can be generatedusing at least one or more of peptide sequences in Table 1: SEQ ID:1-34.

Alternatively and additionally, the inventors also contemplate that theamino sequences of V_(H) segments and/or V_(L) segments presented inTable 1 can be used to generate a recombinant, isolated antibody orfragment thereof. As used herein, the term “antibody” refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain anantigen-binding site that immune-specifically bind an antigen. Thus,“antibody and the fragment thereof” includes a whole immunoglobulinmolecule (e.g., a full-size, whole IgG₁, etc.), a fragment of the wholeantibody molecule. Thus, the fragment thereof may include, but notlimited to, scFv, Fab fragments, Fab′ fragments, F(ab')2, disulfidelinked Fvs (sdFvs), Fvs, and any fragment comprising either V_(H)segment and/or V_(L) segment. Where the antibody is an immunoglobulin,it is contemplate that the immunoglobulin can include any type (e.g.,IgG, IgE, IgM, IgD, IgA and IgY) and any class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) of heavy chain or constant domain to constitutedifferent types of immunoglobulin. In addition, the “antibody” caninclude, but not limited to a human antibody, a humanized antibody, achimeric antibody, a monoclonal antibody, a polyclonal antibody. Thus,it should be appreciated that the V_(H) and/or V_(L) domains as shown inTable 1 can be grafted into any existing (typically human or humanized)antibody or antibody fragment using methods well known in the art.

The inventors further contemplated that the that the amino sequences ofV_(H) segments and/or V_(L) segments presented in Table 1 can be usedcoupled with a carrier protein to generate a hybrid protein having theV_(H) segments and/or V_(L) segments on its surface such that IL-8 canbe captured by the hybrid protein. Any suitable form of carrier proteinthat can stably carry V_(H) segments and/or V_(L) segments andpreferably provide an access to the tumor microenvironment iscontemplated. One especially preferred carrier protein includes albumin,refolded albumin, and other proteins with affinity to antibody portions(e.g., protein A, protein G, protein Z) coupled with one or more V_(H)segments and/or V_(L) segments.

Typically, V_(H) segments and/or V_(L) segments are coupled with ananchor molecule by which V_(H) segments and/or V_(L) segments can becoupled with the carrier protein. For example, where the carrier proteinis an albumin, the anchor molecule can be a hydrophobic peptide orglycolipids in any suitable size to fit in one of Sudlow's site I and IIof the albumin or any other hydrophobic area of the albumin. Forexample, the recombinant immunoglobulin protein against IL-8 asdescribed above can be coupled with the carrier protein via its Fcdomain. In other embodiments, the anchor molecule may include ahydrophobic peptide (in a length of at least 10 amino acids, 15 aminoacids, 20 amino acids, 30 amino acids, etc.). In these embodiments,various configurations of V_(H) segments and/or V_(L) segments (e.g., asa form of scFv) and hydrophobic peptides can be contemplated. Forexample, a monovalent scFv domain can be directly linked to ahydrophobic peptide, or a multivalent scFv can be directly linked to ahydrophobic peptide. Alternatively, one scFv domain can be directlylinked to a plurality of hydrophobic peptides or a plurality of scFvdomain can be directly linked to a plurality of hydrophobic peptides.

Alternatively, or additionally, one or more V_(H) segments and/or V_(L)segments can be coupled with an intermediate molecule that has an anchorportion to bind to the carrier protein. In a preferred embodiment, theinventors contemplate that the intermediate molecule provides aplurality of binding sites for V_(H) segments and/or V_(L) segments suchthat multiple target recognition domains can be carried via a singlebinding site on the carrier protein. Suitable intermediate molecule mayinclude any protein, glycolipid, organic molecule, or inorganic moleculethat does not provide any significant toxicity to the naïve tissue. Forexample, the suitable intermediate molecule may include a nanoparticle(e.g., quantum dots, gold nanoparticles, magnetic nanoparticles,nanotubes, polymeric nanoparticles, dendrimers, etc.), or a bead (e.g.,polystyrene bead, latex bead, dynabead, etc.). Preferably, thenanoparticle and/or beads have a dimension below 1 μm, preferably below100 nm. The nanoparticle may be crosslinked to or partially coated witha hydrophobic tail that provide an anchor to the carrier protein (e.g.,albumin). One or more V_(H) segments and/or V_(L) segments can be alsocrosslinked to or partially coated on the nanoparticles (e.g., via anextra tail domain linked to the target recognition domain forcrosslinking, etc.).

In another example, suitable intermediate molecules may include beads(e.g., polystyrene beads, latex beads, dynabeads, etc.) coupled with anantibody against the carrier protein. Thus, where the carrier protein isan albumin, the beads can be coupled (e.g., crosslinked, coated, etc.)with α-albumin antibody such that the bead can bind to the carrierprotein with a high affinity and specificity. One or more V_(H) segmentsand/or V_(L) segments can be also crosslinked to or partially coated onthe bead (e.g., via an extra tail domain linked to the targetrecognition domain for crosslinking, thiol-mediated crosslinking, etc.).

In some embodiments, scFv peptide can form a recombinant immunoglobulinprotein complex that comprises or mimics an ALT-803 (IL-15 superagonistcomplex, see e.g., Blood 2015 126:1957) or TxM (targeted ALT-803-basedscaffold platform, see e.g., URLaltorbioscience.com/our-science/i1-15-protein-superagonist-and-scaffold-technology/)structure. Preferably, the inventors contemplate that, where theimmunoglobulin protein complex mimics TxM structure, the scFv peptidecan be directly (or indirectly via a linker) coupled to one or moreinterleukin-15 (IL-15) binding motif, and/or one or more ligand to theIL-15 binding motif (e.g., IL-15, IL-15N72D, etc.). Viewed from adifferent perspective, the scFv peptide may be coupled to a proteinhaving IL-15 activity or to a protein having IL-15 receptor alphaactivity. Thus, where the recombinant immunoglobulin protein complexmimics a TxM IgG₁ structure, the recombinant immunoglobulin proteincomplex may include 1-4 scFv peptides against IL-8.

Additionally, the recombinant immunoglobulin protein complex mimickingTxM structure having one or more scFv peptide coupled with IL-15 bindingmotif or its ligand may also include a binding domain against a tumorspecific antigen or patient- and tumor specific neoepitope (e.g., anscFv peptide against the neoepitope, etc). For example, the recombinantimmunoglobulin protein complex may include two scFv peptides againstIL-8 coupled with two IL-15 binding motives and two ScFv peptidesagainst the neoepitope coupled with two IL-15 binding motif ligands.Preferably, the neoepitope is patient-specific and tumor-specific, whichis identified by omics analysis of the sequence data as disclosed in US2012/0059670A1 and US 2012/0066001A1.

In some embodiments, the scFv, recombinant antibody or fragment thereof,can be generated using one sequence encoding a V_(H) segment among SEQID No. 1-15, 31-32. In other embodiments, the scFv, recombinant antibodyor fragment thereof, can be generated using one sequence encoding aV_(L) segment among SEQ ID No. 16-30, 33-34. In still other embodiments,the scFv, recombinant antibody or fragment thereof, can be generatedusing one sequence encoding a V_(H) segment among SEQ ID No. 1-15, 31-32and one sequence encoding a V_(L) segment among SEQ ID No. 16-30, 33-34.In these embodiments, it is preferred that the sequence encoding theV_(H) segment and sequence encoding the V_(L) segment are the paired one(e.g., SEQ ID NO. 1 and 16 for scFv 49-31, SEQ ID NO. 2 and 17 for scFv49-22, SEQ ID NO. 3 and 18 for scFv 49-7, etc.). However, it iscontemplated that any pair of V_(H) segment and V_(L) segment can begenerated by selecting one V_(H) segment from SEQ ID No. 1-15, 31-32 andone V_(L) segment from SEQ ID No. 16-30, 33-34.

TABLE 1  SEQ ID No. Seq name Amino Acid Sequences 1 49-31 V_(H)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMHWVRQAPGKGLEWVSAISGGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLRIFEG RDAGFDVWGQGTLVTVS2 49-22 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLDMFV PFWPAFDVWGQGTLVTVSS3 49-7 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISNSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAENTAVYYCARDLMRW WIDGFDVWGQGTLVTVSS 449-32 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAIYGNGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLYTIEP REAFDVWGQGTLVTVSS5 49-34 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTLGSYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRDTVSI EVGFDVWGQGTLVTVSS6 49-18 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISWSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLYAG YWGFDVWGQGTLVTVSS 749-3 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGKGLEWVSVISGSGGSTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRKFPYVQFFRNGFDVWGQGTLVTVSS 8 43-2 V_(H)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSAISGSGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDREAPFYTVYVTGFDVWGQGTLVTVSS 9 49-37 V_(H)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLWLG WWGFDVWGQGTLVTVSS 1043-12 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYLMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDREGWF WHSYGFDVWGQGTLVTVSS11 49-10 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLGWLS RLFAGFDVWGQGTLVTVSS12 49-1 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWVSAISWSGGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLTWW VQAFDVWGQGTLVTVSS 1349-6 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSEYAMHWVRQAPGKGLEWVSAIDGNGTSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRDTAL ETGFDVWGQGTLVTVSS14 49-12 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMHWVRQAPGKGLEWVSAISNSGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLWWL PAFDVWGQGTLVTVSS 1549-25 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISWSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLYDG VWGFDVWGQGTLVTVSS 1649-31 V_(L) DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDDDVPWTFGQGTKVEIK 17 49-22 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQASDTPLTFGQGTKVEIK 18 49-7 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSYDIPLTFGQGTKVEIK 19 49-32 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDYTSPLTFGQGTKVEIK 20 49-34 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDSAFPLTFGQGTKVEIK 21 49-18 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDYGYPLTFGQGTKVEIK 22 49-3 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHTNGPFTFGQGTKVEIK 23 43-2 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDADTPLTFGQGTKVEIK 24 49-37 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNTIPYTFGQGTKVEIK 25 43-12 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQVYSGPWTFGQGTKVEIK 26 49-10 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDYGYPLTFGQGTKVEIK 27 49-1 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQVDDSPLTFGQGTKVEIK 28 49-6 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQDNAFPLTFGQGTKVEIK 29 49-12 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSSGWPFTFGQGTKVEIK 30 49-25 V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSSYPFTFGQGTKVEIK 31 43-12a V_(H)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYLMHWVRQAPGKGLEWVSAISGSGGITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDREGWF WHSYGFDVWGQGTLVTVSS32 43-12b V_(H) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYLM HWVRQAPGKGLEWVSAISGSGGITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDREGWF WHSYGFDVWGQGTLVTVSS33 43-12a V_(L) DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQVYSGPWTFGQGTKVEIK 34 43-12b V_(L)DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQTYSGPWTFGQGTKVEIK

The inventors also contemplate that the scFv, recombinant antibody orfragment thereof, can be generated with amino acid sequences encodingV_(H) segment and/or V_(L) segment that are at least 85% identical,preferably at least 90% identical, more preferably at least 95%identical to any one of SEQ ID No. 1-15, 31-32 or to any one of SEQ IDNo. 16-30, 33-34, respectively. In such embodiment, it is preferred thatthe binding affinity of the scFv peptide, recombinant antibody orfragment thereof, is no less than 60%, preferably no less than 70%, morepreferably no less than 80% of binding affinity of a scFv peptide,recombinant antibody or fragment thereof, generated with any one of SEQID No. 1-15, 31-32 or to any one of SEQ ID No. 1-15, 31-32. Indeed, andas discussed in further detail below, the inventors used a selected scFv(43-12) and subjected the scFv to affinity maturation via randommutagenesis in the CDR regions for V_(H) and V_(L). Notable, and amongother binders, two scFv (43-12a and 43-12b) were isolated from theaffinity maturation process that had improved binding characteristics.

Most typically, the V_(H) segment and V_(L) segment in scFv peptide,recombinant antibody or fragment thereof, are coupled via a linker or aspacer, which is typically between 5-40 amino acids, preferably between10-30 amino acids, more preferably between 20-30 amino acids. In someembodiments, the linker can couple the N-terminus of V_(H) segment andC-terminus of V_(L) segment. In other embodiments, the linker can couplethe N-terminus of V_(L) segment and C-terminus of V_(H) segment. Theinventors contemplate that glycine-rich sequences (e.g., (G₄S)_(n), withn between 1-5, etc.) for the linker are preferred to provide structuralflexibility between the V_(H) segment and V_(L) segments. It is alsocontemplated that the linker includes one or more serine or threonineresidue to increase solubility of the scFv peptide, recombinant antibodyor fragment thereof. There are numerous linkers and methods of makingscFv known in the art, and all such known methods are deemed suitablefor use herein.

Additionally, the scFv peptide can include a plurality of V_(H) segmentsand V_(L) segments to form a divalent or multivalent scFv. In someembodiments, the plurality of V_(H) segments and/or V_(L) segments mayhave same amino sequences (e.g., a multivalent scFv having three V_(H)segments and three V_(L) segments, and all V_(H) segments have 49-31V_(H) (SEQ ID. No.1) and all V_(L) segments have 49-31 V_(L) (SEQ ID.No.16). In other embodiments, at least two of V_(H) segments and/orV_(L) segments may have different amino acid sequences (e.g., amultivalent scFv having three V_(H) segments and three V_(L) segments,and two of V_(H) segments are 49-31 V_(H) (SEQ ID. No.1) and one ofV_(H) segments is 49-22 V_(H) (SEQ ID. No.2), and two of V_(L) segmentsare 49-31 V_(L) (SEQ ID. No.16) and one of V_(H) segments is 49-22 V_(L)(SEQ ID. No. 17).

Preferably, the binding affinity (Kd) of the scFv, recombinant antibodyor fragment thereof, to IL-8 (at least one of 72-mer and 77-mer IL-8) isat least less than 1×10⁻⁷M, preferably less than 1×10⁻⁸M, and morepreferably less than 1×10⁻⁹M, measured at a temperature between 25° C.to 37° C., and measured in a pH range between 5.5-7.5. The inventorscontemplate that the binding affinities of the scFv, recombinantantibody or fragment thereof, to IL-8 may be different due to thestructural differences even if the scFv, recombinant antibody orfragment thereof, are generated using the same amino acid sequences ofV_(H) segment and/or V_(L) segments. For example, Table 2 providesdifferent affinities (measured in KD value) of scFv and recombinantantibody (IgG1) of several clones. Clone 49-31 is a scFv or arecombinant antibody (IgG1) generated using amino acid sequencesencoding V_(H) segment (SEQ ID No. 1) and V_(L) segment (SEQ ID No. 16),Clone 43-2 is a scFv or a recombinant antibody (IgG1) generated usingamino acid sequences encoding V_(H) segment (SEQ ID No. 8) and V_(L)segment (SEQ ID No. 23), and Clone 43-12 is a scFv or a recombinantantibody (IgG1) generated using amino acid sequences encoding V_(H)segment (SEQ ID No.10) and V_(L) segment (SEQ ID No. 25). PreliminaryK_(D) values are determined by surface plasmon resonance measurement,and highest and lowest values are shown to estimate the range of theaffinities to IL-8. All unit of Kd value in Table 2 is 10⁻⁹ M.

TABLE 2 IgG1 IgG1 IgG1 (25° C., (25° C., (37° C., Clone scFv pH 7.4) pH6.0) pH 7.4) 49-31 0.009-0.048 0.009-0.25 0.21-0.22 0.117 43-2  1.410.79 0.30-0.96 0.72-0.89 43-12 1.50  0.43-0.78 3.46-6.7  0.31 

Table 3 provides further exemplary data for molecules having sequencesas shown in Table 1. Here, scFv and corresponding humanized IgG₁ wereprepared using the sequences as indicated, and KD was determined usingSPR with immobilized IL-8 as the analyte on the chip surface. All valuesare expressed as nanoM values at temperatures as indicated.

TABLE 3 scFv IgG1 IgG Clone (25° C.) (25° C.) (37° C.) 49-31 0.009-0.0480.099-0.25  0.12 49-22  0.11-0.114 49-7  0.087-0.17  49-32 0.21-0.240.77-0.87 0.33-0.36 49-34 0.44-0.62 49-18 0.41-0.89 49-3  1.26 2.14 9.1943-2  1.41 0.79 0.72-0.89 49-37 1.12-1.46 43-12 1.50 0.43-0.78 0.3149-10 1.65 2.21 3.45 49-1  2.66 49-6  4.8  49-12 10.1  49-25 25.0 

It is further contemplated that the scFv peptide (or V_(H) segment andV_(L) segment of an antibody or fragment thereof) can be encoded by asingle recombinant nucleic acid. In this embodiment, the recombinantnucleic acid includes at least two nucleic acid segments: a firstnucleic acid segment (a sequence element) encoding a V_(H) segment and asecond nucleic acid segment encoding a V_(L) segment. It is preferredthat the first nucleic acid segment is selected to encode at least oneof amino acid sequences of SEQ ID. No 1-15, 31-32, and the secondnucleic acid segment is selected to encode at least one of amino acidsequences of SEQ ID. No 16-30, 33-34. However, it is also contemplatedthat the first and second nucleic acid segment encode V_(H) and V_(L)segment respectively that are at least 85% identical, preferably atleast 90% identical, more preferably at least 95% identical to any oneof SEQ ID No. 1-15, 31-32 or to any one of SEQ ID No. 16-30, 33-34. Mostpreferably, the two nucleic acid segments are in the same reading framesuch that two nucleic acid segments can be translated into a singleprotein having two peptide segments.

Additionally, the recombinant nucleic acid may include a third nucleicacid segment between the first and second nucleic acid segment encodinga linker peptide (preferably G-rich or otherwise flexible linkerpeptide), which is typically between 5-40 amino acids, preferablybetween 10-30 amino acids, more preferably between 20-30 amino acids. Inthis embodiment, it is especially preferred that the three nucleic acidsegments are in the same reading frame such that three nucleic acidsegments can be translated into a single protein having three peptidesegments.

In some embodiments, the recombinant nucleic acid may include aplurality of sets of first and second nucleic acid segments, in whicheach set includes one of each first and second nucleic acid segment. Inthese embodiments, the recombinant nucleic acid preferably includes afourth nucleic acid segment encoding a connector peptide located betweeneach set of the first and second nucleic acid segments. Thus, oneexemplary recombinant nucleic acid may include [first set]-fourthnucleic acid encoding a connector peptide-[second set]-fourth nucleicacid encoding the connector peptide-[third set], where each of first,second, and third set includes [a first nucleic acid encoding V_(H)segment-a third nucleic acid encoding a linker-a second nucleic acidencoding V_(L) segment], where the location of the first and secondnucleic acid can be alternated with each other. The sequence of theconnector peptide may vary depending on the number of sets in a singlepeptide. Preferably, the connector peptide may be between 5-50 aminoacids, preferably between 10-40 amino acids, more preferably between20-30 amino acids. Also inventors contemplates that glycine-richsequences (e.g., G₄S, etc.) are preferred to provide flexibility of theconnector peptide between the V_(H) and V_(L) segment sets.

The inventors further contemplate that the scFv peptide, antibodies orfragments thereof, can be formulated as a pharmaceutical composition sothat it can be administered to the patient having a tumor to reduce orinhibit the endogenous effect of IL-8. Therefore, it is contemplatedthat the scFv peptide, antibodies or fragments thereof, can beformulated in any pharmaceutically acceptable carrier (e.g., as asterile injectable composition) in an amount of at least 1 ml,preferably at least 5 ml, more preferably and at least 20 ml per dosageunit for a therapeutic formulation. However, alternative formulationsare also deemed suitable for use herein, and all known routes and modesof administration are contemplated herein. As used herein, the term“administering” refers to both direct and indirect administration of thecompounds and compositions contemplated herein, where directadministration is typically performed by a health care professional(e.g., physician, nurse, etc.), while indirect administration typicallyincludes a step of providing or making the compounds and compositionsavailable to the health care professional for direct administration.

In some embodiments, the pharmaceutical formulation is administered viasystemic injection including subcutaneous, subdermal injection, orintravenous injection. In other embodiments, where the systemicinjection may not be efficient (e.g., for brain tumors, etc.), it iscontemplated that the formulation is administered via intratumoralinjection.

One exemplary method and use of pharmaceutical composition includingscFv peptide, the antibody or fragment thereof, which includes V_(H) andV_(L) segments described above is to reduce IL-8 mediated effects in atarget tissue. As used herein, the IL-8 mediated effect refers to anybiological consequence induced directly or indirectly by presence ofIL-8 in the tissue or the microenvironment of the tissue. Thus, the IL-8effect may be originated from IL-8 released (or secreted) by a tumorcell and/or a non-tumor cell in the tissue (e.g., immune competent cellsuch as lymphocytes etc.) or outside of the tissue (e.g., a healthytissue near the tumor, etc.) and present at any given time in the tumormicroenvironment.

In particularly contemplated embodiments, the inventors contemplate thatthe IL-8 mediated effect, especially in the tumor microenvironment of apatient, includes, but is not limited to, triggering transmigration of acell, triggering epithelial-mesenchymal transition of the tumor,triggering metastasis of the tumor, increasing immune suppression in thetumor microenvironment, stimulation of MDSC development, and triggeringTh2-biased immune response in the tumor microenvironment. Thus, andwithout wishing to be bound to specific theory, the inventorscontemplate that reducing the quantity of IL-8 from the tumormicroenvironment by trapping free IL-8 (IL-8 not bound to IL-8 receptor)reduces the IL-8 effect or even reverse the IL-8 effect to the tumorcells: reducing epithelial-mesenchymal transition of the tumor, reducingor preventing metastasis of the tumor, reducing Th2-biased immuneresponse or rebalancing Th1- and Th2- mediated immune response, andreducing or preventing immune suppression in the tumor microenvironment.Especially, where excessive or abnormal increase of IL-8 expression (oraccumulation) is shown in specific types of cancers (e.g., pancreaticcancer, triple negative breast cancer, glioblastoma, etc.) relative tocorresponding healthy tissue, it is expected that binding or entrapmentof IL-8 may change the prognosis of those types of cancers moreeffectively.

With respect to dose and schedule of the pharmaceutical compositionadministration to a patient, it is contemplated that the dose and/orschedule may vary depending on the type of peptides (e.g., scFv, anantibody, an antibody fragment, combination of any two of those,combination of all, etc.), type and prognosis of disease (e.g., tumortype, size, location), health status of the patient (e.g., includingage, gender, etc.). While it may vary, the dose and schedule may beselected and regulated so that the formulation does not provide anysignificant toxic effect to the host normal cells, yet sufficient to bereduce IL-8 effect in the tumor microenvironment at least 20%,preferably at least 30%, more preferably at least 40%, most preferablyat least 50% within less than 3 hours, 6 hours, 12 hours, 24 hours, 72hours, or a week.

In some embodiments, the effect of IL-8 and the reduction of IL-8 effectcan be measured by quantity of free IL-8 (either 72-mer or 77-mer) inthe tissue. In these embodiments, the administration conditions aretypically adjusted to have a quantity or concentration of at least oneof 72-mer or 77-mer free IL-8 reduced at least 30%, more preferably atleast 40%, most preferably at least 50% within less than 3 hours, 6hours, 12 hours, 24 hours, 72 hours, or a week after administering thepharmaceutical composition. In other embodiments, the effect of IL-8 andthe reduction of IL-8 effect can be measured via in vitro or in vivomeasurement of biological activities. For example, the dose andtreatment schedule for reducing cell migration or metastasis of a tumorcan be determined by in vitro cell migration assay by IL-8. In thisexample, the administration conditions are typically adjusted to reducethe number of cells migrating from the original position or to reducethe distance of migration of migrating cells at least 20%, preferably atleast 30%, more preferably at least 40%, most preferably at least 50%within 1 hour, 3 hours, 6 hours, 12 hours after treatment with thepharmaceutical composition (or scFv, antibody or fragment thereofwithout pharmaceutically acceptable carrier). For other example, thedose and treatment schedule for reducing immune suppression bymyeloid-derived suppressor cells can be determined by measuring theaccumulation or presence of myeloid-derived suppressor cells in thetumor. Thus, in this example, the administration conditions aretypically adjusted to reduce the number of myeloid-derived suppressorcells (in the entire tumor tissue or per cm² of tumor tissue) at least20%, preferably at least 30%, more preferably at least 40%, mostpreferably at least 50% within 3 hours, 6 hours, 12 hours, 24 hours, 72hours, or a week after administering the pharmaceutical composition.

In still other embodiments, the effect of IL-8 and the reduction of IL-8effect can be measured via measuring quantity or concentration of localcytokine molecules. For example, the dose and treatment schedule forreducing Th-2 mediated immune response can be determined by measuringthe quantity or concentration of at least one of IL-4, IL-5, IL-6, IL-9,IL-19, and IL-13 in the tumor microenvironment. Thus, in this example,the administration conditions are typically adjusted to reduce thequantity or concentration of at least one of IL-4, IL-5, IL-6, IL-9,IL-19, and IL-13 for at least 20%, preferably at least 30%, morepreferably at least 40%, most preferably at least 50% within 3 hours, 6hours, 12 hours, 24 hours, 72 hours, or a week after administering thepharmaceutical composition.

In still other embodiments, the effect of IL-8 and the reduction of IL-8effect can be measured via measuring, in vitro or in vivo, expressionlevel of one or more cellular markers. For example, the dose andtreatment schedule for reducing epithelial-mesenchymal transition oftumor cells can be determined by measuring expression of E-cadherinepithelial marker and N-cadherin mesenchymal marker in the tumor cells.Thus, the administration conditions are typically adjusted to reduce theexpression level of N-cadherin mesenchymal marker at least 20%,preferably at least 30%, more preferably at least 40%, most preferablyat least 50% within 3 hours, 6 hours, 12 hours, 24 hours, 72 hours, or aweek after administering the pharmaceutical composition, or increase theexpression level (compared to IL-8 treatment only) of E-cadherinepithelial marker at least 20%, preferably at least 30%, more preferablyat least 40%, most preferably at least 50% within 3 hours, 6 hours, 12hours, 24 hours, 72 hours, or a week after administering thepharmaceutical composition. It is also contemplated that the ratiobetween N-cadherin and E-cadherin expression in the tumor cells can bealso be an indicator of the effect of IL-8 and the reduction of IL-8effect. In this example, the administration conditions are typicallyadjusted to increase the ratio of E-cadherin: N-cadherin expressionlevels at least 20%, preferably at least 30%, more preferably at least40%, most preferably at least 50% within 3 hours, 6 hours, 12 hours, 24hours, 72 hours, or a week after administering the pharmaceuticalcomposition.

Viewed from a different perspective, the reduction of IL-8 mediatedeffects in vivo can also be observed by physiological phenomena. Forexample, reduced IL-8 concentration can be observed via reduction orabolishment of EMT (epithelial-mesenchymal transition) of tumor cellsand reduction in the associated signaling pathways. Similarly, reducedIL-8 concentrations in a patient and especially in a tumormicroenvironment will reduce the sternness of tumor cells as can bereadily observed by appropriate stem cell markers. In yet otherphysiological effects of IL-8 reduction, development of MDSC istypically reduced, as well as a Th2 biased immune response in T cellswithin the tumor (thus shifting the Th1/Th2 balance towards a Th1 typeresponse.

Additionally, the inventors contemplate that the effect of scFv,antibody or fragment thereof against IL-8 to reduceepithelial-mesenchymal transition of tumor cells in specific type ofcancer can be boosted by co-administration of one or more cancermedications. The cancer medications includes, but not limited to,Fulvestrant, Aldoxorubicin, Docetaxel, a tumor necrosis treatment agent(e.g., ¹³¹I-chTNT-3, etc.), Avelumab (a human monoclonal IgG₁ antibodythat blocks interaction between PD-L1 and its receptor),Brachyury-targeting vaccine (e.g., ETBX-051 (Ad5 [E1-,E2b-]-Brachyury)), Her2-targeting vaccine (e.g., ETBX-021, etc.),MUC-1-targeting vaccine (e.g., ETBX-061 (Ad5 [E1-, E2b-]-MUC1)), andyeast vaccines (e.g., GI-4000 (GI-4014, GI-4015, GI-4016, GI-4020),GI-6207, GI-6301). Details of these cancer medications are described inPCT/US17/40297, which is incorporated herein as a reference in itsentirety.

In some embodiments, the inventors contemplate that the effect of scFv,antibody or fragment thereof against IL-8 can be boosted withco-administration of one or more checkpoint inhibitors. With respect toa protein that interferes with or down-regulates checkpoint inhibition,it is contemplated any suitable peptide ligands that bind to acheckpoint receptor are contemplated. Most typically, binding willinhibit or at least reduce signaling via the receptor, and particularlycontemplated receptors include CTLA-4 (especially for CD8⁺ cells), PD-1(especially for CD4⁺ cells), TIM1 receptor, 2B4, and CD160. For example,suitable peptide binders can include antibody fragments and especiallyscFv, but also small molecule peptide ligands (e.g., isolated via RNAdisplay or phage panning) that specifically bind to the receptors. Oncemore, it should be appreciated that expression of the peptide moleculeswill preferably be coordinated such that the neoepitopes or polytope areexpressed contemporaneously with one or more of the peptide ligands.Thus, it is typically contemplated that the peptide ligands are producedfrom a single transcript (which may or may not include the sequenceportion encoding the polytope), for example, using an internal ribosomeentry site or 2A sequence, or from multiple transcripts.

In other embodiments, the inventors contemplate that one or moreimmune-stimulatory cytokines can be co-administered to modulate theeffect of scFv, antibody or fragment thereof against IL-8. For example,the immune stimulatory cytokines can be selected based on the desiredimmune response or direction(s) of CD4+ T cell/naive Th cellpolarization. For example, in an embodiment where polarization of Tregcells from naive CD4+T cells is desired, the immune stimulatory cytokinemay be selected to include IL-2 and TGF-β. In another embodiment wherepolarization of Th17 cells from naive CD4+T cells is desired, the immunestimulatory cytokine may be selected to include IL-6 and TGF-β.Likewise, the immune stimulatory cytokine for Th1 cell polarization mayinclude IL-12 and IFN-γ, and the immune stimulatory cytokine for Th2cell polarization may include IL-4. Additionally, the immune stimulatorycytokine for Tfh cell (follicular helper T cell) polarization mayinclude IL-6 and IL-12, and the immune stimulatory cytokine for CD4+cytotoxic T cell polarization may include IL-2.

The inventors further contemplate that the scFv, antibody or fragmentthereof against IL-8 can be co-treated (or co-administered) to thecancer patient with activated or modified immune cells that can providethe scFv, antibody or fragment thereof against IL-8, better access tothe tumor microenvironment by reducing the effect of immune suppressionby tumor cells or myeloid-derived suppressor cells in the tumormicroenvironment so that the effect of the scFv, antibody or fragmentthereof against IL-8 can be maximized. For example, the activated ormodified immune cells may include naive NKT cells or geneticallymodified NKT cells expressing a chimeric protein or T cell receptorcomplex to induce NKT cell immune response, and/or to change themicroenvironment of the tumor (e.g., by suppressing activity ofmyeloid-derived suppressor cells). Preferably, the chimeric protein or Tcell receptor of genetically modified NKT cells binds a tumor(neo)epitope, a tumor associated antigen, or a self-lipid presented onthe tumor cells. For other example, the modified immune cells mayinclude NKT cells genetically modified to express at least one of CD40Land Fas-L, preferably on their cell surfaces. Details of geneticallymodified and/or naive, activated NKT cells to reduce immune suppressionin the cancer microenvironment are described in Int. App. No.PCT/US18/53506 (and its corresponding published US national phaseapplication), which is incorporated herein in its entirety. Likewise,the activated or modified immune cells may include naive T cells orgenetically modified T cells expressing a chimeric protein or T cellreceptor complex to induce T cell immune response, and/or to change themicroenvironment of the tumor (e.g., by suppressing activity ofmyeloid-derived suppressor cells). Preferably, the chimeric protein or Tcell receptor of genetically modified NKT cells binds a tumor(neo)epitope, a tumor associated antigen, or a self-lipid presented onthe tumor cells.

For still other example, the activated or modified immune cells mayinclude genetically modified NK cells expressing killer activatingreceptor (KAR) targeting soluble NK cell receptor ligands (e.g., NKG2D,Nkp-30, Nkp-44, Nkp-46, etc), which prevents effective NK cell activityby acting as a decoy ligand to the NK cell receptors. Details ofgenetically modified NK cells with KAR are described in U.S. Prov. App.No. 62/569503, which is incorporated herein in its entirety.

In some embodiments, one or more above cancer medications, immunestimulatory cytokines, checkpoint inhibitors, and/or naïve orgenetically modified NK cells or NKT cells can be formulated in the samepharmaceutical composition with scFv, antibody or fragment thereof,against IL-8. In other embodiments, the cancer medication can formulatedin a separate pharmaceutical composition that can be provided togetherwith the pharmaceutical composition of scFv, antibody or fragmentthereof, or administered before the patient is administered with thepharmaceutical composition of scFv, antibody or fragment thereof

Therefore, it should be appreciated that the IL-8 binding moleculespresented herein may form of a treatment regimen that includesadministration of the IL-8 binding molecules presented herein. Forexample, it is contemplated that treatment of a cancer patient (e.g.,pancreatic cancer, triple negative breast cancer, glioblastoma) may beperformed in an orchestrated manner in which a first drug (e.g.,fulvestrant) is administered to reverse EMT to MET (mesenchymal toepithelial transition) and in which a second drug (e.g., aldoxorubicin)is administered to specifically target the hypoxic tumormicroenvironment. Such treatment regimen is thought to reduce immunesuppressive environment and increase cell stress in a tumor cell,resulting in an increased immunogenicity of the tumor cells to theimmune system. Moreover, the treatment may also include tumor necrosistargeting antibodies that will ‘label’ necrotic tumor cells and soincrease susceptibility to attack by NK cells and/or cytotoxic T cells.The IL-8 binding molecules presented herein are co-administered tofurther suppress IL-8 mediated immune suppressive effects as notedabove. Additionally, upon reduction of immune suppressive conditions asnoted above, the patient may then receive immune therapy, typicallyusing recombinant vaccines that express one or more of brachyury, tumoror cancer associated antigens, and/or patient- and tumor specificneoantigens. Thus, cancer treatment will include at least two (or atleast three, or at least four) of the following: A tumor necrosistargeting drug, a drug that targets the hypoxic tumor microenvironment,a drug that reverses EMT to MET, a vaccine component (recombinant virus,yeast, and/or bacteria), and IL-8 binding molecules as presented herein.

EXAMPLES

The inventors generated scFv molecules (43-12) using nucleic acidsequences of SEQ ID No. 10 (43-12 V_(H)) encoding V_(H) segment and SEQID No. 25 (43-12 V_(L)) encoding V_(L) segment, and determined thebinding affinities to IL-8 (72-mer and 77-mer) and cross-reactivities toother orthologs or paralogs of IL-8. As shown in FIG. 1A, the 43-12 scFvmolecule exhibits strong affinity to both 72-mer and 77-mer of IL-8(with KD 385 pM and 440 pM), and very little cross reactivities, if any,with paralogs (hCXCL1, hCXCL2, hCXCL7) or an ortholog (mCXCL1).

The inventors also generated scFv molecules (49-31) using nucleic acidsequences of SEQ ID No. 1 (49-31 V_(H)) encoding V_(H) segment and SEQID No. 16 (49-31 V_(L)) encoding V_(L) segment, and determined thebinding affinities to IL-8 (72-mer and 77-mer) and cross-reactivities toother orthologs or paralogs of IL-8. As shown in FIG. 1B, the 49-31 scFvmolecule exhibits strong affinity to both 72-mer and 77-mer of IL-8(with KD 147 pM and 120 pM), and very little cross reactivities, if any,with paralogs (hCXCL1, hCXCL2, hCXCL7) or an ortholog (mCXCL1).

Next, the inventors tested so-generated scFv molecules (43-2, 43-12,49-31, etc. all also shown in Table 3) to determine their neutralizingeffect of IL-8. In one exemplary set of experiments, primary humanneutrophils were isolated from blood to assess the ability of the scFvmolecules (here: 43-2, 43-12, 49-31) to neutralize IL-8 mediatedneutrophil chemotaxis. In a 3 mM pore size transwell plate, 1 nM ofrecombinant human IL-8 was incubated with a titration of antibodies asindicated in the left panel, and 50,000 calcein-AM labeled neutrophilswere added into the top chamber for 1.5 and 3 hours. Transmigration ofneutrophils was assessed by assaying for total fluorescence in thebottom chamber and background subtracted against blank media only wells.FIG. 2, panels A-D show graphs representing neutrophil migration invitro upon treatment of IL-8 alone (FIG. 2A, FIG. 2C) and IL-8 and oneof scFv molecules (43-2, 43-12, 49-31) for 1.5 hours (FIG. 2B) or for 3hours (FIG. 2D). As shown in FIG. 2B and 2D, all scFv molecules (43-2,43-12, 49-31) could reduce the transmigration of neutrophils 30-70% withless than 1 uM concentration in both 1.5 hours and 3 hours, indicatingthat all scFv molecules (43-2, 43-12, 49-31) are effective in mitigatingthe effect of IL-8 by entrapping free IL-8 in the serum (or media).

FIG. 3 shows a graph indicating the neutrophil transmigration upontreatment of IL-8 alone (CTRL) and IL-8 and one of scFv molecules (43-2,43-12) in 1 nM concentration. Both scFv molecules (43-2, 43-12) couldcompletely reverse the effect of IL-8 such that the levels of neutrophilmigration with scFv molecules (43-2, 43-12) treatment are similar oralmost identical to media alone, while IL-8 treatment alone couldincrease the neutrophil migration almost three times compared to themedia alone.

In yet another set of experiments, the inventors used affinitymaturation (here: random mutagenesis of CDR regions in VH and VL chainsand mRNA display selection) of one previously identified IL-8 binder(here: 43-12, sequence in Table 1), and SPR analysis was performed onselected mutant forms using two different pH conditions, pH 7.4 and pH6.0. As can be seen from the results in FIGS. 4A-C (with FIG. 4Adepicting results for the parent scFv 43-12), both derivative mutantforms had improved binding over the parent scFv. Specifically, FIG. 4Bshows exemplary results for 43-12a (sequences shown in Table 1) and FIG.4C shows exemplary results for 43-12b (sequences shown in Table 1).

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. As used in the description herein and throughoutthe claims that follow, the meaning of “a,” “an,” and “the” includesplural reference unless the context clearly dictates otherwise. Also, asused in the description herein, the meaning of “in” includes “in” and“on” unless the context clearly dictates otherwise. Where thespecification claims refers to at least one of something selected fromthe group consisting of A, B, C . . . and N, the text should beinterpreted as requiring only one element from the group, not A plus N,or B plus N, etc.

What is claimed is:
 1. A method of treating a patient having a tumor,comprising: providing a pharmaceutical composition including arecombinant antibody or fragment thereof or single chain variablefragment (scFv) peptide, wherein the antibody, fragment thereof, or scFvpeptide bind to IL-8; wherein the antibody, fragment thereof, or scFvpeptide has a V_(H) segment comprising a first amino acid sequenceselected from the group consisting of SEQ ID NO. 1-15, 31-32, and aV_(L) segment comprising a second amino acid sequence selected from thegroup consisting of SEQ ID NO. 16-30, 33-34, to thereby generate theantibody, fragment thereof or scFv peptide; and administering thepharmaceutical composition to the patient.
 2. The method of claim 1,wherein the V_(H) segment and V_(L) segment are coupled to each other bya linker peptide to thereby form the scFv peptide.
 3. The method ofclaim 2, wherein the linker peptide is glycine-rich peptide.
 4. Themethod of claim 1 wherein the scFv peptide has a V_(H) segmentcomprising an amino acid sequence having SEQ ID NO. 1, and a V_(L)segment comprising an amino acid sequence having SEQ ID NO. 16, orwherein the scFv peptide has a V_(H) segment comprising an amino acidsequence having SEQ ID NO. 2, and a V_(L) segment comprising an aminoacid sequence having SEQ ID NO. 17, or wherein the scFv peptide has aV_(H) segment comprising an amino acid sequence having SEQ ID NO. 3, anda V_(L) segment comprising an amino acid sequence having SEQ ID NO. 18,or wherein the scFv peptide has a V_(H) segment comprising an amino acidsequence having SEQ ID NO. 4, and a V_(L) segment comprising an aminoacid sequence having SEQ ID NO. 19, or wherein the scFv peptide has aV_(H) segment comprising an amino acid sequence having SEQ ID NO. 5, anda V_(L) segment comprising an amino acid sequence having SEQ ID NO. 20,or wherein the scFv peptide has a V_(H) segment comprising an amino acidsequence having SEQ ID NO. 6, and a V_(L) segment comprising an aminoacid sequence having SEQ ID NO. 21, or wherein the scFv peptide has aV_(H) segment comprising an amino acid sequence having SEQ ID NO. 7, anda V_(L) segment comprising an amino acid sequence having SEQ ID NO. 22,or wherein the scFv peptide has a V_(H) segment comprising an amino acidsequence having SEQ ID NO. 8, and a V_(L) segment comprising an aminoacid sequence having SEQ ID NO. 23, or wherein the scFv peptide has aV_(H) segment comprising an amino acid sequence having SEQ ID NO. 9, anda V_(L) segment comprising an amino acid sequence having SEQ ID NO. 24,or wherein the scFv peptide has a V_(H) segment comprising an amino acidsequence having SEQ ID NO. 10, and a V_(L) segment comprising an aminoacid sequence having SEQ ID NO. 25, or wherein the scFv peptide has aV_(H) segment comprising an amino acid sequence having SEQ ID NO. 11,and a V_(L) segment comprising an amino acid sequence having SEQ ID NO.26, or wherein the scFv peptide has a V_(H) segment comprising an aminoacid sequence having SEQ ID NO. 12, and a V_(L) segment comprising anamino acid sequence having SEQ ID NO. 27, or wherein the scFv peptidehas a V_(H) segment comprising an amino acid sequence having SEQ ID NO.13, and a V_(L) segment comprising an amino acid sequence having SEQ IDNO. 28, or wherein the scFv peptide has a V_(H) segment comprising anamino acid sequence having SEQ ID NO. 14, and a V_(L) segment comprisingan amino acid sequence having SEQ ID NO. 29, or wherein the scFv peptidehas a V_(H) segment comprising an amino acid sequence having SEQ ID NO.15, and a V_(L) segment comprising an amino acid sequence having SEQ IDNO. 30, or wherein the scFv peptide has a V_(H) segment comprising anamino acid sequence having SEQ ID NO. 31, and a V_(L) segment comprisingan amino acid sequence having SEQ ID NO. 33, or wherein the scFv peptidehas a V_(H) segment comprising an amino acid sequence having SEQ ID NO.32, and a V_(L) segment comprising an amino acid sequence having SEQ IDNO.
 34. 5. The method of claim 1, wherein the scFv peptide is selectedfrom the group consisting of 43-2, 43-12, 49-1, 49-3, 49-6, 49-7, 49-10,49-12, 49-18, 49-22, 49-25, 49-31, 49-32, 49-34, and 49-37.
 6. Themethod of claim 1, wherein the pharmaceutical composition includes therecombinant antibody.
 7. The method of claim 6 wherein the recombinantantibody has a V_(H) segment comprising an amino acid sequence havingSEQ ID NO. 1, and a V_(L) segment comprising an amino acid sequencehaving SEQ ID NO. 16, or wherein the recombinant antibody has a V_(H)segment comprising an amino acid sequence having SEQ ID NO. 2, and aV_(L) segment comprising an amino acid sequence having SEQ ID NO. 17, orwherein the recombinant antibody has a V_(H) segment comprising an aminoacid sequence having SEQ ID NO. 3, and a V_(L) segment comprising anamino acid sequence having SEQ ID NO. 18, or wherein the recombinantantibody has a V_(H) segment comprising an amino acid sequence havingSEQ ID NO. 4, and a V_(L) segment comprising an amino acid sequencehaving SEQ ID NO. 19, or wherein the recombinant antibody has a V_(H)segment comprising an amino acid sequence having SEQ ID NO. 5, and aV_(L) segment comprising an amino acid sequence having SEQ ID NO. 20, orwherein the recombinant antibody has a V_(H) segment comprising an aminoacid sequence having SEQ ID NO. 6, and a V_(L) segment comprising anamino acid sequence having SEQ ID NO. 21, or wherein the recombinantantibody has a V_(H) segment comprising an amino acid sequence havingSEQ ID NO. 7, and a V_(L) segment comprising an amino acid sequencehaving SEQ ID NO. 22, or wherein the recombinant antibody has a V_(H)segment comprising an amino acid sequence having SEQ ID NO. 8, and aV_(L) segment comprising an amino acid sequence having SEQ ID NO. 23, orwherein the recombinant antibody has a V_(H) segment comprising an aminoacid sequence having SEQ ID NO. 9, and a V_(L) segment comprising anamino acid sequence having SEQ ID NO. 24, or wherein the recombinantantibody has a V_(H) segment comprising an amino acid sequence havingSEQ ID NO. 10, and a V_(L) segment comprising an amino acid sequencehaving SEQ ID NO. 25, or wherein the recombinant antibody has a V_(H)segment comprising an amino acid sequence having SEQ ID NO. 11, and aV_(L) segment comprising an amino acid sequence having SEQ ID NO. 26, orwherein the recombinant antibody has a V_(H) segment comprising an aminoacid sequence having SEQ ID NO. 12, and a V_(L) segment comprising anamino acid sequence having SEQ ID NO. 27, or wherein the recombinantantibody has a V_(H) segment comprising an amino acid sequence havingSEQ ID NO. 13, and a V_(L) segment comprising an amino acid sequencehaving SEQ ID NO. 28, or wherein the recombinant antibody has a V_(H)segment comprising an amino acid sequence having SEQ ID NO. 14, and aV_(L) segment comprising an amino acid sequence having SEQ ID NO. 29, orwherein the recombinant antibody has a V_(H) segment comprising an aminoacid sequence having SEQ ID NO. 15, and a V_(L) segment comprising anamino acid sequence having SEQ ID NO. 30, or wherein the recombinantantibody has a V_(H) segment comprising an amino acid sequence havingSEQ ID NO. 31, and a V_(L) segment comprising an amino acid sequencehaving SEQ ID NO. 33, or wherein the recombinant antibody has a V_(H)segment comprising an amino acid sequence having SEQ ID NO. 32, and aV_(L) segment comprising an amino acid sequence having SEQ ID NO.
 34. 8.The method of claim 6 wherein the recombinant antibody is selected fromthe group consisting of 49-31, 43-2, 43-12, 49-32, 49-3, and 49-10. 9.The method of claim 1, wherein the pharmaceutical composition includesthe recombinant antibody fragment.
 10. The method of claim 9 wherein therecombinant antibody fragment is coupled to a protein having IL-15activity or to a protein having IL-15 receptor alpha activity.
 11. Themethod of claim 1, wherein the scFv peptide is coupled to a proteinhaving IL-15 activity or to a protein having IL-15 receptor alphaactivity.
 12. The method of claim 1, further comprising a step ofadministering at least one of the following: Fulvestrant, Aldoxorubicin,Docetaxel, a tumor necrosis treatment agent (TNT), and a tumor vaccine.13. The method of claim 1, wherein the antibody, fragment thereof, orscFv peptide has a binding affinity to IL-8 of equal or less than 10⁻⁷M.
 14. The method of claim 1, further comprising co-administering to thepatient an immune-stimulatory cytokine selected from a group consistingof IL-2, IL-12, IL-15, an IL-15 super agonist (ALT803), IL-21, IPS1, andLMP1.
 15. The method of claim 1, wherein the pharmaceutical compositionis administered in a dose and a schedule effective to treat the tumor.16. The method of claim 1, wherein the pharmaceutical composition isadministered in a dose and a schedule effective to reduce free IL-8 inthe tumor in the patient by at least 30%.
 17. The method of wherein thepharmaceutical composition is administered in a dose and a scheduleeffective to block epithelial-mesenchymal transition of the tumor in thepatient by at least 30%.
 18. The method of wherein the pharmaceuticalcomposition is administered in a dose and a schedule effective to reducethe number of myeloid-derived suppressor cells in the tumor in thepatient by at least 30%.
 19. The method of claim 1, wherein the tumor isat least one of the following: pancreatic cancer, triple negative breastcancer, glioblastoma.
 20. The method of claim 1, further comprisingco-administering to the patient a checkpoint inhibitor, or a tumorvaccine.